Time-lapse Optical Coherence Tomography Research Articles

High-sensitivity synchronous angio-lymphography based on a speckle spectrum contrast OCT.

To achieve accurate selection and synchronous imaging of blood vessels and lymph, a speckle spectrum contrast method (SSC) based on spectral-domain optical coherence tomography (SD-OCT) is proposed in this Letter. In this method, the time-lapse optical coherence tomography (OCT) intensity signal is transformed to the Fourier frequency domain. By analyzing the frequency spectrum of the time-lapse OCT intensity signal, a parameter called SSC signal, which represents the ratio of different intervals of the high frequency to the low frequency, is utilized to extract and contrast different types of the vessels in the biological tissues. In the SSC spectrum, the SSC signals of the static tissue, lymphatic vessels, and vascular vessels can be separated in three different frequency intervals, enabling differentiation and synchronous imaging of the lymphatic-vascular vessels. A mouse ear was used to demonstrate the feasibility and efficiency of this method. By using the SSC signal as the imaging parameter, the lymphatic and blood vessels of the mouse ear are differentiated and visualized simultaneously. This study shows the feasibility of the three-dimensional (3D) synchronous angio-lymphography based on the SSC method, which provides a tool to improve the understanding for disease research and treatment.

P-295 Application of a Time-Lapse Optical Coherence Tomography (OCT) approach in a pilot study to visualise oocytes and embryos in depth

Abstract Study question Can we visualise model oocytes and embryos during embryo development using OCT non-invasively? Summary answer This approach, new to embryology, OCT, allows visualising cells in a 3D perspective, providing more detailed information than standard microscopy from the oocyte or embryo. What is known already Time-lapse is a well-established technique in human IVF, but it is limited in its depth of view due to the limitations of classical microscopy. To overcome this limitation, the approach of Optical Coherence Tomography (OCT), allows non-invasive visualisation through optical cross-sections of the embryo or oocyte to produce 3D images. The obtained information can be extended to 4 or 5 dimensions to track movement and elasticity, using a low-power light source and no staining to ensure the minimum effect on the cell. Here, a pilot study was performed in model (porcine) samples using a newly developed “in incubator” system. Study design, size, duration A pilot basic study was performed where 16 oocytes and 16 cleavage embryos were obtained and prepared to be visualised using OCT. Participants/materials, setting, methods Pig ovaries were obtained from a slaughterhouse, from which follicles were aspirated in order to retrieve oocytes. The best-quality oocytes were cultured for maturation for 44 hours and 16 oocytes were selected for OCT imaging. before fertilisation for 2 hours. Sperm was previously prepared by Percoll gradient. Zygotes were cultured for 6 days until visualisation through OCT. For imaging, samples were prepared in a 16-well Primo Vision dish. Main results and the role of chance Oocytes and embryos were successfully imaged, allowing the identification of distinct cellular features. In oocytes, it was possible to identify the germinal vesicle (nucleus) and polar bodies, individual blastomeres in cleavage stage embryos and trophectoderm, ICM and blastocoel in blastocysts. As images were taken throughout different optical sections, it was possible to correlate the position of the areas within the oocytes/embryos and create a 3D image from a blastocyst, understanding the size of the inner cell mass compared with the embryo's overall size. Interestingly, images were obtained non-invasively and under optimal conditions (in an incubator), demonstrating the future utility of OCT for embryo imaging. Limitations, reasons for caution An experimental OCT system was developed and placed in the incubator, and all images obtained were in a format of a pilot study. Future imaging sessions are planned to obtain more data points and assess the viability of the embryo. Wider implications of the findings Although in the present study, OCT was applied in pig oocytes and embryos, this new approach can be employed in future for human IVF to overcome the current imaging limitations, proving more information on the oocytes and embryos’ quality and assisting Artificial Intelligence analysis. Trial registration number not applicable

Frequency-constrained robust principal component analysis: a sparse representation approach to segmentation of dynamic features in optical coherence tomography imaging

Sparse representation theory is an exciting area of research with recent applications in medical imaging and detection, segmentation, and quantitative analysis of biological processes. We present a variant on the robust-principal component analysis (RPCA) algorithm, called frequency constrained RPCA (FC-RPCA), for selectively segmenting dynamic phenomena that exhibit spectra within a user-defined range of frequencies. The algorithm lacks subjective parameter tuning and demonstrates robust segmentation in datasets containing multiple motion sources and high amplitude noise. When tested on 17 ex-vivo, time lapse optical coherence tomography (OCT) B-scans of human ciliated epithelium, segmentation accuracies ranged between 91-99% and consistently out-performed traditional RPCA.

Time-Series Interactions of Gene Expression, Vascular Growth and Hemodynamics during Early Embryonic Arterial Development.

The role of hemodynamic forces within the embryo as biomechanical regulators for cardiovascular morphogenesis, growth, and remodeling is well supported through the experimental studies. Furthermore, clinical experience suggests that perturbed flow disrupts the normal vascular growth process as one etiology for congenital heart diseases (CHD) and for fetal adaptation to CHD. However, the relationships between hemodynamics, gene expression and embryonic vascular growth are poorly defined due to the lack of concurrent, sequential in vivo data. In this study, a long-term, time-lapse optical coherence tomography (OCT) imaging campaign was conducted to acquire simultaneous blood velocity, pulsatile micro-pressure and morphometric data for 3 consecutive early embryonic stages in the chick embryo. In conjunction with the in vivo growth and hemodynamics data, in vitro reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to track changes in transcript expression relevant to histogenesis and remodeling of the embryonic arterial wall. Our non-invasive extended OCT imaging technique for the microstructural data showed continuous vessel growth. OCT data coupled with the PIV technique revealed significant but intermitted increases in wall shear stress (WSS) between first and second assigned stages and a noticeable decrease afterwards. Growth rate, however, did not vary significantly throughout the embryonic period. Among all the genes studied, only the MMP-2 and CASP-3 expression levels remained unchanged during the time course. Concurrent relationships were obtained among the transcriptional modulation of the genes, vascular growth and hemodynamics-related changes. Further studies are indicated to determine cause and effect relationships and reversibility between mechanical and molecular regulation of vasculogenesis.

Progressive Early Breakdown of Retinal Pigment Epithelium Function in Hyperglycemic Rats.

PurposeDiabetic macular edema (DME), an accumulation of fluid in the subretinal space, is a significant cause of vision loss. The impact of diabetes on the breakdown of the inner blood–retina barrier (BRB) is an established event that leads to DME. However, the role of the outer BRB in ocular diabetes has received limited attention. We present evidence that the breakdown of normal RPE function in hyperglycemia facilitates conditions conducive to DME pathogenesis.MethodsBrown Norway rats (130–150 g) were injected intraperitoneally with streptozotocin (STZ; 60 mg/kg) to induce hyperglycemia. After 4 weeks, Evans blue (EB) dye was injected intravenously to determine whether there was leakage of albumin into the retina. Subretinal saline blebs (0.5–1 μL) were placed 4 and 9 weeks after STZ injection, and time-lapse optical coherence tomography tracked the resorption rate. In a subset of rats, intravitreal bevacizumab, a humanized monoclonal antibody targeted to VEGF, was given at 5 weeks and resorption was measured at 9 weeks.ResultsThe ability of the RPE to transport fluid was reduced significantly after 4 and 9 weeks of hyperglycemia with a reduction of over 67% at 9 weeks. No EB dye leakage from inner retinal vessels was measured in hyperglycemic animals compared to control. The intravitreal administration of bevacizumab at week 5 significantly increased the rate of fluid transport in rats subjected to hyperglycemia for 9 weeks.ConclusionsThese results demonstrate that chronic hyperglycemia altered RPE fluid transport, in part dependent on the actions of VEGF. These results support the idea that RPE dysfunction is an early event associated with hyperglycemia that contributes to fluid accumulation in DME.

Volumetric monitoring of aqueous two phase system droplets using time-lapse optical coherence tomography

We present a volumetric monitoring method to observe the morphological changes of aqueous two phase system (ATPS) droplets in a microfluidic system. Our method is based on time-lapse optical coherence tomography (OCT) which allows the study of the dynamics of ATPS droplets while visualizing their 3D structures and providing quantitative information on the droplets. In this study, we monitored the process of rehydration and deformation of an ATPS droplet in a microfluidic system and quantified the changes of its volume and velocity under both static and dynamic fluid conditions. Our results indicate that time-lapse OCT is a very promising tool to evaluate the unprecedented features of droplet-based microfluidics.

Receptor mediated disruption of retinal pigment epithelium function in acute glycated-albumin exposure

Diabetic macular edema (DME) is a major cause of visual impairment. Although DME is generally believed to be a microvascular disease, dysfunction of the retinal pigment epithelium (RPE) can also contribute to its development. Advanced glycation end-products (AGE) are thought to be one of the key factors involved in the pathogenesis of diabetes in the eye, and we have previously demonstrated a rapid breakdown of RPE function following glycated-albumin (Glyc-alb, a common AGE mimetic) administration in monolayer cultures of fetal human RPE cells. Here we present new evidence that this response is attributed to apically oriented AGE receptors (RAGE). Moreover, time-lapse optical coherence tomography in Dutch-belted rabbits 48 h post intravitreal Glyc-alb injections demonstrated a significant decrease in RPE-mediated fluid resorption in vivo. In both the animal and tissue culture models, the response to Glyc-alb was blocked by the relatively selective RAGE antagonist, FPS-ZM1 and was also inhibited by ZM323881, a relatively selective vascular endothelial growth factor receptor 2 (VEGF-R2) antagonist. Our data establish that the Glyc-alb-induced breakdown of RPE function is mediated via specific RAGE and VEGF-R2 signaling both in vitro and in vivo. These results are consistent with the notion that the RPE is a key player in the pathogenesis of DME.

The use of time-lapse optical coherence tomography to image the effects of microapplied toxins on the retina.

We developed a novel technique for accelerated drug screening and retinotoxin characterization using time-lapse optical coherence tomography (OCT) and a drug microapplication device. Using an ex vivo rabbit eyecup preparation, we studied retinotoxin effects in real-time by microperfusing small retinal areas under a transparent fluoropolymer tube. Known retinotoxic agents were applied to the retina for 5-minute periods, while changes in retinal structure, thickness, and reflectance were monitored with OCT. The OCT images of two agents with dissimilar mechanisms, cyanide and kainic acid, were compared to their structural changes seen histologically. We found the actions of retinotoxic agents tested could be classified broadly into two distinct types: (1) agents that induce neuronal depolarization, such as kainic acid, causing increases in OCT reflectivity or thickness of the inner plexiform and nuclear layers, and decreased reflectivity of the outer retina; and (2) agents that disrupt mitochondrial function, such as cyanide, causing outer retinal structural changes as evidenced by a reduction in the OCT reflectivity of the photoreceptor outer segment and pigment epithelium layers. Retinotoxin-induced changes in retinal layer reflectivity and thickness under the microperfusion tube in OCT images closely matched the histological evidence of retinal injury. Time-lapse OCT imaging of the microperfused local retina has the potential to accelerate drug retinotoxicological screening and expand the use of OCT as an evaluation tool for preclinical animal testing.

Label-Free, Longitudinal Visualization of PDT Response In Vitro with Optical Coherence Tomography.

A major challenge in creating and optimizing therapeutics in the fight against cancer is visualizing and understanding the microscale spatiotemporal treatment response dynamics that occur in patients. This is especially true for photodynamic therapy (PDT), where therapeutic optimization relies on understanding the interplay between factors such as photosensitizer localization and uptake, in addition to light dose and delivery rate. In vitro 3D culture systems that recapitulate many of the biological features of human disease are powerful platforms for carrying out detailed studies on PDT response and resistance. Current techniques for visualizing these models, however, often lack accuracy due to the perturbative nature of the sample preparation, with light attenuation complicating the study of intact models. Optical coherence tomography (OCT) is an ideal method for the long-term, non-perturbative study of in vitro models and their response to PDT. Monitoring the response of 3D models to PDT by time-lapse OCT methods promises to provide new perspectives and open the way to cancer treatment methodologies that can be translated towards the clinic.

Early Detection of Carcinoma In Situ of the Bladder: A Comparative Study of White Light Cystoscopy, Narrow Band Imaging, 5-ALA Fluorescence Cystoscopy and 3-Dimensional Optical Coherence Tomography

We compared the efficacy and potential limitations of white light cystoscopy, narrow band imaging, 5-ALA fluorescence cystoscopy and 3-dimensional optical coherence tomography for early diagnosis of bladder carcinoma in situ. By expressing simian virus 40T antigen in the urothelium carcinoma in situ typically develops in SV40T transgenic mice in about 8 to 20 weeks and then frank high grade papillary urothelial carcinoma starts to emerge. A total of 18 control and 29 SV40T mice were examined during weeks 8 to 22 by white light cystoscopy, fluorescence cystoscopy, narrow band imaging and 3-dimensional optical coherence tomography. Results were validated by histology. Newly improved algorithms for computer aided detection were applied to acquired 3-dimensional optical coherence tomography images to enhance the quantitative diagnosis of carcinoma in situ in near real time. Of 29 carcinoma in situ samples 27 were detected by 3-dimensional optical coherence tomography, 1 by white light cystoscopy, 26 by narrow band imaging and 13 by fluorescence cystoscopy. Of the 18 histologically confirmed benign cases 17 were detected by 3-dimensional optical coherence tomography, 14 by white light cystoscopy, 5 by narrow band imaging and 18 by fluorescence cystoscopy. The diagnostic sensitivity of white light cystoscopy (3.4%) and fluorescence cystoscopy (44.8%), and the specificity of narrow band imaging (27.8%) were significantly enhanced by 3-dimensional optical coherence tomography to 93.1% and 94.4%, respectively (p <0.01). Three-dimensional optical coherence tomography with quantitative computer aided detection can significantly enhance the sensitivity of white light cystoscopy and fluorescence cystoscopy, and the specificity of narrow band imaging for early diagnosis of carcinoma in situ. This suggests the potential of narrow band imaging guided 3-dimensional optical coherence tomography for future clinical detection of carcinoma in situ when effective image guidance is desirable.

Killing Hypoxic Cell Populations in a 3D Tumor Model with EtNBS-PDT

An outstanding problem in cancer therapy is the battle against treatment-resistant disease. This is especially true for ovarian cancer, where the majority of patients eventually succumb to treatment-resistant metastatic carcinomatosis. Limited perfusion and diffusion, acidosis, and hypoxia play major roles in the development of resistance to the majority of front-line therapeutic regimens. To overcome these limitations and eliminate otherwise spared cancer cells, we utilized the cationic photosensitizer EtNBS to treat hypoxic regions deep inside in vitro 3D models of metastatic ovarian cancer. Unlike standard regimens that fail to penetrate beyond ∼150 µm, EtNBS was found to not only penetrate throughout the entirety of large (>200 µm) avascular nodules, but also concentrate into the nodules' acidic and hypoxic cores. Photodynamic therapy with EtNBS was observed to be highly effective against these hypoxic regions even at low therapeutic doses, and was capable of destroying both normoxic and hypoxic regions at higher treatment levels. Imaging studies utilizing multiphoton and confocal microscopies, as well as time-lapse optical coherence tomography (TL-OCT), revealed an inside-out pattern of cell death, with apoptosis being the primary mechanism of cell killing. Critically, EtNBS-based photodynamic therapy was found to be effective against the model tumor nodules even under severe hypoxia. The inherent ability of EtNBS photodynamic therapy to impart cytotoxicity across a wide range of tumoral oxygenation levels indicates its potential to eliminate treatment-resistant cell populations.

Abstract 3261: Visualizing treatment response dynamics of an in vitro three-dimensional ovarian cancer model

Abstract Ovarian epithelial cancer (OVCA) is the fifth leading cause of cancer death among women in the US. The majority of patients have advanced-stage disseminated OVCA that coats the many surfaces in the abdomen with widespread nodular disease. These metastatic lesions often become drug resistant following standard chemotherapy regiments, leading to an unacceptably low survival rate of about 30%. In order to more effectively eradicate this metastatic disease, it is imperative to understand of the dynamics of ovarian tumor treatment response at the cellular and nodular levels. A three-dimensional in vitro model of OVCA developed in our group has proven to be a promising system for visualizing and optimizing the therapy of ovarian nodular disease, enabling studies that would otherwise be implausible to carry out in vivo. Ovarian cancer cells plated on Matrigel spontaneously self-assemble to form a heterogeneous mixture of three-dimensional spheroids that replicate many of the molecular and morphological features observed in avascular micrometastatic nodules found in vivo. Using the non-perturbative three-dimensional structural imaging technology known as optical coherence tomography (OCT), we have observed these nodules to grow beyond 500 µm in diameter and display complex structural changes during development. We have used this model system as a platform for multimodal OCT and fluorescence microscopy experiments aimed at understanding the treatment response of ovarian micronodular disease to cytotoxic therapies. High-throughput confocal fluorescence viability imaging alongside time-lapse OCT experiments revealed that treatments with the front-line chemotherapeutics cisplatin and carboplatin do not effectively kill cells in the center of large (&amp;gt;300 µm diameter) nodules. This three-dimensional pattern of cell death suggests a possible treatment resistance mechanism in vivo. To target this seemingly resistant cell population, we have used photodynamic therapy (PDT), an alternative modality that has been found to be effective against platinum-resistant ovarian cancer. PDT utilizes molecules known as a photosensitizers that, when exposed to a particular wavelength of light, generate cytotoxic reactive oxygen and radical species. We found that treatment using the photosensitizer EtNBS results in effective cellular killing of the previously spared nodule cores. Moreover, when combined with carboplatin, we observed a synergistic treatment response compared to the individual monotherapies alone. Ongoing studies using the 3D model aim to examine the underlying mechanisms behind these treatment responses and will be important in optimizing new therapeutic regimens for metastatic ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3261.

Subcellular imaging of epithelium with time-lapse optical coherence tomography

We present the first experimental result of direct delineation of the nuclei of living rat bladder epithelium with ultrahigh-resolution optical coherence tomography (uOCT). We demonstrate that the cellular details embedded in the speckle noise in a uOCT image can be uncovered by time-lapse frame averaging that takes advantage of the micromotion in living biological tissue. The uOCT measurement of the nuclear size (7.9+/-1.4 microm) closely matches the histological evaluation (7.2+/-0.8 microm). Unlike optical coherence microscopy (OCM), which requires a sophisticated high-NA microscopic objective, this approach uses a commercial-grade single achromatic lens (f/10 mm, NA/0.25) and provides a cross-sectional image over 0.6 mm of depth without focus tracking, thus holding great promise of endoscopic optical biopsy for diagnosis and grading of flat epithelial cancer such as carcinoma in situ in vivo.

High-resolution imaging characterization of bladder dynamic morphophysiology by time-lapse optical coherence tomography

We report an experimental study of the possibility of high-speed optical coherence tomography (OCT) for high-resolution imaging characterization of detrusor dynamic morphophysiology and analysis of the mechanisms that lead to geriatric incontinence (GI). The spontaneous contractility of intact fresh rabbit bladders was imaged with two-dimensional (2D) OCT ex vivo at up to 8 frames/s. The time-lapse 2D OCT images were postprocessed by image segmentation and fast-Fourier-transform analysis to characterize the dynamic morphological changes of the bladder contractility. In addition, we studied young and aging rat bladders to analyze the differences in dynamics. Preliminary results of our ex vivo study reveal that time-lapse OCT can track the contractile waves of bladders at high spatial resolution and characterize their dynamic morphophysiology in terms of amplitude, phase, and frequency. The results suggest that time-lapse OCT has the potential to act as a detrusor optical biopsy to enhance the diagnosis of detrusor dysfunction and thus of the mechanisms that lead to GI.